Objective: The water-soluble extract of Ganoderma lucidum mycelia (WER), which is used as a health food, reduced hyperglycemia and enhanced glucose transporter 4 (GLUT4) translocation to the plasma membrane in skeletal muscle and adipose tissue in KK-A^y mice, a type 2 diabetic animal model with obesity.In order to elucidate the reduction of hyperglycemia by WER, we investigated the translocation of glucose transporter 4, glucose uptake and associating signal transduction in rat skeletal muscle (L6) cells. Method: The glucose uptake was analyzed with radioactive 2-deoxy-D-glucose.The localization of GLUT4 in L6 cells treated with various concentrations of WER was analyzed with immunohistochemical staining and Western blot technique.As a positive control, insulin or troglitazone was used in these experiments.Furthermore, the activation of intracellular signaling pathways by Western blot analysis and the influence of glucose uptake using four kinds of inhibitors (LY294002 as potent PI3K inhibitor, rapamycin as mTOR inhibitor, Gö6983 as broad PKC inhibitor, compound C as AMPK inhibitor) was evaluated. Results: GLUT4 protein content in the plasma membrane was induced in a dose-dependent manner of WER without increasing the gene expression and amount of total protein in the L6 cells and the glucose uptake was augmented with increasing the amount of GLUT4 translocated on the plasma membrane.The phosphorylation of phosphatidylinositol-3 kinase (PI3K), Akt and acetyl CoA carboxylase (ACC) were induced in a concentration dependent manner and inhibited by the above inhibitors except rapamycin. Conclusion: These results indicate that the hypoglycemic effect of some material(s) in WER may be due to the enhancement of glucose uptake through GLUT4 translocation on the plasma membrane by activating the PI3K/Akt pathway through improving insulin resistance.